CN111018698A - Monomer compound separated from Fritillaria pallidiflora extract and preparation method and application thereof - Google Patents

Monomer compound separated from Fritillaria pallidiflora extract and preparation method and application thereof Download PDF

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CN111018698A
CN111018698A CN201911357392.6A CN201911357392A CN111018698A CN 111018698 A CN111018698 A CN 111018698A CN 201911357392 A CN201911357392 A CN 201911357392A CN 111018698 A CN111018698 A CN 111018698A
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fritillaria pallidiflora
silica gel
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CN111018698B (en
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石磊岭
马国需
魏鸿雁
金悦仙
马晓玲
阿勒腾图娅
关永强
宋海龙
陈刚
郭雄飞
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XINJIANG UIGHUR AUTONOMOUS REGION CHINESE MEDICINE NATIONAL DRUG INSTITUTE
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Abstract

The invention relates to the technical field of separation and purification of Fritillaria pallidiflora, and a preparation method and application thereof, and discloses monomeric compounds separated from Fritillaria pallidiflora.

Description

Monomer compound separated from Fritillaria pallidiflora extract and preparation method and application thereof
Technical Field
The invention relates to the technical field of separation and purification of Fritillaria pallidiflora, and relates to a monomer compound separated from Fritillaria pallidiflora extract and a preparation method and application thereof.
Background
Fritillaria pallidiflora Schrenk is a Fritillaria plant of Liliaceae Fritillaria, mainly distributed in the Yili river basin of Xinjiang, and mainly produced in Yining, Shosu, Suiudin, Hodgkin, Nile, Borle and other places in the northwest of Xinjiang. The bulb is used as a medicine, is bitter, sweet and slightly cool in taste, enters lung and heart channels, has the effects of relieving cough, eliminating phlegm and relieving asthma clinically, has the effects of calming and resisting anoxia, is very favorable for asthma patients, and has the effects of diminishing inflammation and resisting bacteria to treat respiratory tract infection cooperatively. The Fritillaria pallidiflora is a special drug in Xinjiang, is one of traditional vitamins, has high yield, has higher alkaloid content compared with similar Fritillaria pallidiflora, strong disease resistance and low price of medicinal materials, particularly has slightly stronger physiological activities of relieving cough, eliminating phlegm, resisting inflammation, resisting bacteria and the like than the Fritillaria unibracteata, and because the Fritillaria unibracteata resource is gradually exhausted in recent years, the research on alternative varieties of the Fritillaria unibracteata is continuously carried out by people, the artificial cultivation of the Fritillaria pallidiflora is realized and can be popularized and planted in a large area, so the Fritillaria unibracteata pallidiflora.
The drug effect of the Fritillaria pallidiflora is mainly derived from steroid alkaloid compounds, but the reports of non-alkaloid compounds are rare, so that the non-alkaloid monomeric compounds of the Fritillaria pallidiflora are developed and utilized, the potential medicinal value of the non-alkaloid monomeric compounds is further excavated, and the structure and the physicochemical properties of the monomeric compounds are determined and characterized, thereby having important significance for developing and utilizing the Fritillaria pallidiflora.
Disclosure of Invention
The invention provides a monomer compound separated from an extract of Fritillaria pallidiflora pall, a preparation method and an application thereof, and discloses the monomer compound separated from the extract of Fritillaria pallidiflora pall, 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxy-.
One of the technical schemes of the invention is realized by the following measures that the monomer compounds separated from the extract of Fritillaria pallidiflora pall comprise 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybutyl-1-alkenyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Saiwannin-6-oxo-5 α,20 β -diol ibeiannin, wherein the chemical structural formula of the 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid is shown in the specification
Figure BDA0002336294560000011
Chemistry of 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanolStructural formula is
Figure BDA0002336294560000021
The chemical structural formula of the 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol is shown in the specification
Figure BDA0002336294560000022
The chemical structural formula of the 17 β -Seawanine-6-oxo-5 α,20 β -diol ebeibenine is shown in the specification
Figure BDA0002336294560000023
The following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:
the monomer compound separated from the Fritillaria pallidiflora extract is obtained by the following steps of firstly, crushing Fritillaria pallidiflora, adding methanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, carrying out 1-3 hours each time, merging refluxing extracting solutions each time, carrying out reduced pressure recovery and concentration to obtain Fritillaria pallidiflora total extract, secondly, adjusting the pH value of the Fritillaria pallidiflora total extract to 3.5 by using 2% hydrochloric acid, carrying out kneading and filtering to obtain acid liquor, carrying out extraction degreasing on the acid liquor by using petroleum ether, adjusting the pH value to 10 by using ammonia water, carrying out extraction by using chloroform, concentrating the extracting solution to obtain chloroform part extract, thirdly, carrying out gradient elution and separation on the chloroform part by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient of the silica gel column chromatography comprises petroleum ether, acetone and triethylamine, the volume ratio of the petroleum ether, the acetone and the triethylamine is sequentially 20: 1: 0.1: 1: 1.1: 1: 1.1, the fraction and the triethylamine, carrying out gradient elution on the silica gel column chromatography, the gradient chromatography to the silica gel column chromatography to the gradient elution to the silica gel column chromatography to the gradient chromatography to the silica gel column chromatography to the silica gel chromatography to obtain the methanol fraction, the methanol fraction is subjected to the methanol fraction, the methanol fraction is subjected to the methanol fraction, the methanol fraction is subjected to the methanol fraction, the methanol fraction.
In the first step, 8ml to 10ml of methanol is added to 1g of Fritillaria pallidiflora.
In the fourth step, the fifth step, the sixth step and the seventh step, the eluent for gradient elution of the high performance liquid chromatography is a mixed solution of methanol and water, and the volume ratio of the methanol to the water in the mixture is 60: 40. 70: 30. 85: 15 and 80: 20.
the second technical scheme is that the preparation method of the monomer compound separated from the Fritillaria pallidiflora extract is implemented through the following steps of firstly, crushing Fritillaria pallidiflora, adding methanol, soaking for 3 hours to 4 hours at room temperature, heating and refluxing for 3 times at 50 ℃ to 60 ℃ for 1 hour to 3 hours each time, combining refluxing extract liquid for each time, decompressing and recovering, concentrating to obtain Fritillaria pallidiflora total extract, secondly, adjusting pH of the Fritillaria pallidiflora total extract to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid liquid, extracting and degreasing the acid liquid with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extract liquid to obtain chloroform extract, thirdly, performing gradient elution and separation on the chloroform extract to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography comprises petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether to acetone to triethylamine is 20: 1: 0.1: 1: 0.5, performing gradient elution and separation on the chloroform extract to 1: 1.5, performing gradient elution for 1: 1: 2 to 5, purifying on the eluent to 5-7 th, performing gradient elution, collecting the phenyl group chromatography to 2 to obtain a hydroxyl group, performing gradient elution, purifying on the eluent to obtain a fourth step, performing gradient elution to 3-2, performing gradient elution to obtain a phenyl group chromatography, and purification, and collecting the fourth step, and purifying on the fourth step, and collecting the phenyl group III, and purifying on the eluent to obtain the third, and purifying on the fourth step, the phenyl group III fraction, the fourth step, the phenyl group of the eluent, the fourth step, the phenyl group of the fourth step, the.
The following is further optimization or/and improvement of the second technical scheme of the invention:
in the first step, 8ml to 10ml of methanol is added to 1g of Fritillaria pallidiflora.
In the fourth step, the fifth step, the sixth step and the seventh step, the eluent for gradient elution of the high performance liquid chromatography is a mixed solution of methanol and water, and the volume ratio of the methanol to the water in the mixture is 60: 40. 70: 30. 85: 15 and 80: 20.
the third technical scheme of the invention is realized by the following measures: an application of a monomer compound separated from Fritillaria pallidiflora extract in preparing medicine for preventing tumor is disclosed.
The fourth technical scheme of the invention is realized by the following measures: an application of a monomer compound separated from Fritillaria pallidiflora extract in preparing antineoplastic agent is provided.
The fifth technical scheme of the invention is realized by the following measures: an application of a monomer compound separated from Fritillaria pallidiflora extract in preparing health products for preventing and treating tumor is disclosed.
The invention discloses a monomeric compound 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybut-1-alkenyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Saiwannin-6-oxo-5 α,20 β -diol ebeibening separated from an extract of Fritillaria pallida for the first time, wherein the monomeric compound has a strong inhibiting effect on HepG-2 cells and HeLa cells, and can be applied to preparation of tumor prevention medicines, anti-tumor medicines and health-care products for preventing and treating tumors.
Drawings
FIG. 1 shows the isolated monomeric compound 7- (4-hydroxy-3-Process for preparation of methoxyphenyl) -9, 10-anhydride-8-propionic acid1H-NMR spectrum.
FIG. 2 shows the monomeric compound 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol isolated from Fritillaria pallidiflora extract1H-NMR spectrum.
FIG. 3 shows the preparation of 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol as a monomeric compound isolated from Fritillaria pallidiflora extract1H-NMR spectrum.
FIG. 4 shows the monomer compound 17 β -Saiwanning-6-oxo-5 α,20 β -diol ibeibenin isolated from Fritillaria pallidiflora extract1H-NMR spectrum.
FIG. 5 shows the isolation of 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid as a monomeric compound from Fritillaria pallidiflora extract13C-APT spectrum.
FIG. 6 shows the monomeric compound 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol isolated from Fritillaria pallidiflora extract13C-APT spectrum.
FIG. 7 shows the isolation of 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol, a monomeric compound, from an extract of Fritillaria pallidiflora13C-APT spectrum.
FIG. 8 shows the monomer compound 17 β -Saiwanning-6-oxo-5 α,20 β -diol ibeibenin isolated from Fritillaria pallidiflora extract13C-APT spectrum.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments may be determined according to the technical solutions and practical situations of the present invention. The various chemical reagents and chemical articles mentioned in the invention are all the chemical reagents and chemical articles which are well known and commonly used in the prior art, unless otherwise specified; the percentages in the invention are mass percentages unless otherwise specified; the solution in the present invention is an aqueous solution in which the solvent is water, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution, unless otherwise specified; the normal temperature and room temperature in the present invention generally mean a temperature of 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to the following examples:
example 1 monomeric compounds isolated from Fritillaria pallidiflora extract, including 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propanoic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol, and 17 β -Savonin-6-oxo-5 α,20 β -diol ibeinnine, wherein 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propanoic acid has the chemical structure
Figure BDA0002336294560000041
The chemical structural formula of the 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol is
Figure BDA0002336294560000042
The chemical structural formula of the 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol is shown in the specification
Figure BDA0002336294560000043
The chemical structural formula of the 17 β -Seawanine-6-oxo-5 α,20 β -diol ebeibenine is shown in the specification
Figure BDA0002336294560000051
Example 2 the monomeric compounds separated from the Fritillaria pallidiflora extract are obtained by the steps of crushing Fritillaria pallidiflora, adding methanol, soaking at room temperature for 3-4 hours, heating and refluxing at 50-60 ℃ for 3 times, recovering under reduced pressure and concentrating to obtain Fritillaria pallidiflora total extract, adjusting pH to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid solution, extracting the acid solution with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extract to obtain chloroform fraction, subjecting the chloroform fraction to gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the gradient chromatography comprises petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether, acetone and triethylamine is 20: 1: 0.1, 15: 1: 0.1, 10: 1: 0.1, 5: 1, 1: 1: 1.1: 1: 1.5, purifying with phenyl-2, purifying with phenyl-3-7, purifying with phenyl-2-3-7-3-2-3-7-3-2-3-2-3-2-7-3-2-3-7-3-2-3-three-2-three.
Example 3: as an optimization of the above example, in the first step, 8ml to 10ml of methanol is added per 1g of Fritillaria pallidiflora.
Example 4: as an optimization of the above embodiment, in the fourth step, the fifth step, the sixth step and the seventh step, eluents for gradient elution by high performance liquid chromatography are all mixed solutions of methanol and water, and the volume ratio of methanol to water in the mixture is 60: 40. 70: 30. 85: 15 and 80: 20.
example 5 the monomer compound separated from the Fritillaria pallidiflora extract is obtained by the steps of adding 8 ml-10 ml of methanol into every 1g of Fritillaria pallidiflora, crushing Fritillaria pallidiflora and adding methanol, soaking for 3 hours to 4 hours at room temperature, heating and refluxing for 3 times at 50 ℃ to 60 ℃ for 1 hour to 3 hours each time, combining refluxing extract liquid each time, recovering under reduced pressure, concentrating to obtain Fritillaria pallidiflora total extract, adjusting pH of Fritillaria pallidiflora total extract to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid liquid, extracting and defatting the acid liquid with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extract to obtain chloroform extract, performing gradient elution on chloroform extract to obtain 8 fractions, wherein the gradient elution of silica gel column chromatography is performed to obtain 8 fractions, the gradient eluent of silica gel column chromatography includes petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether, acetone and triethylamine is 20: 1: 0.1: 1: 0.5, the gradient chromatography is 0.5: 1: 2, the gradient elution is 0.5-5: 2, the gradient elution is performed to 5, the steps of methanol-5, the gradient elution is performed to 5, the steps of the gradient elution is performed to 10, the steps of the gradient chromatography to 5, the gradient elution is performed to 5, the gradient chromatography to 5, the gradient elution is performed to 5, the steps of the gradient chromatography to 5, the gradient elution is performed to 5, the gradient chromatography to 5, the gradient chromatography is performed to 5, the gradient chromatography to 5, the eluent is performed to 5 gradient chromatography to the gradient chromatography to 5, the gradient chromatography to the eluent is performed to the eluent to the.
Example 6 the monomeric compounds separated from the Fritillaria pallidiflora extract are obtained by the steps of adding 10ml of methanol into 1g of Fritillaria pallidiflora, crushing the Fritillaria pallidiflora, adding methanol, soaking for 4 hours at room temperature, heating and refluxing for 3 times and 3 hours at 60 ℃, merging reflux extracting solutions, recovering under reduced pressure, concentrating to obtain Fritillaria pallidiflora total extract, adjusting pH to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid solution, extracting and degreasing the acid solution with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extract to obtain chloroform extract, performing gradient elution with silica gel column chromatography to obtain 8 fractions, wherein the gradient of silica gel column chromatography comprises petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether, acetone and triethylamine is 20: 1: 0.1, 15: 1: 0.1: 1: 0.1: 1: 5: 1: 1.5, the gradient of methanol to 5: 0.5: 1: 2: 5: 2, purifying the eluent with methanol, the gradient of methanol-5, the gradient of methanol-5 gradient to obtain methanol, purifying the gradient of the gradient in the gradient of the gradient to obtain methanol gradient, the gradient of the eluent, and the gradient of the eluent, and the gradient of the eluent is the eluent, and the eluent is the eluent from the fourth gradient of the eluent, the third step of the eluent, the eluent is obtained by the third step of the fourth step of the eluent, the eluent is obtained by the steps of the third step of the fourth step of the eluent is obtained by the eluent, the eluent is the eluent, the eluent is the eluent, the eluent is the eluent, the eluent is the eluent, the.
Example 7 the monomeric compounds separated from the Fritillaria pallidiflora extract are obtained by a first step of adding 8ml of methanol to 1g of Fritillaria pallidiflora, crushing the Fritillaria pallidiflora and adding methanol, soaking for 3 hours at room temperature, heating and refluxing for 3 times at 50 ℃ for 1 hour each time, combining refluxed extract solutions, recovering under reduced pressure, concentrating to obtain Fritillaria pallidiflora total extract, a second step of adjusting the pH value of the Fritillaria pallidiflora total extract to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid solution, extracting and degreasing the acid solution with petroleum ether, adjusting the pH value to 10 with ammonia water, extracting with chloroform, concentrating the extract to obtain chloroform extract, a third step of performing gradient elution and separation on the chloroform extract with a silica gel column chromatography to obtain 8 fractions, wherein the gradient chromatography of the silica gel column comprises petroleum ether, acetone and triethylamine, the volume ratio of the petroleum ether, acetone and triethylamine is 0.1: 1: 0.1: 1: 0.1: 1: 5: 0.1: 2, the volume ratio of the methanol to 5: 2: 5: 2, the gradient chromatography to 5: 5, the gradient chromatography to 5-5 volume ratio of the eluent is obtained by a fifth step of the steps of the third step of the methanol, the gradient chromatography to 5, the eluent is obtained by a third step of the fourth step of the methanol, the fourth step of the methanol to the methanol, the fourth step of the methanol to the third step of the methanol, the eluent is the fourth step of the third step of the methanol, the fourth step of the methanol to the fourth step of the methanol to the fourth step of the third step of the fourth step of the methanol to the fourth step of the fourth.
Example 8 the monomer compound separated from the Fritillaria pallidiflora extract is obtained by adding 9ml of methanol into 1g of Fritillaria pallidiflora, crushing the Fritillaria pallidiflora, adding methanol, soaking for 3.5 hours at room temperature, heating and refluxing for 3 times and 2 hours at 55 ℃, combining refluxing extracting solutions, recovering under reduced pressure, concentrating to obtain Fritillaria pallidiflora total extract, adjusting pH to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid solution, extracting and degreasing the acid solution with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extracting solution to obtain chloroform extract, performing silica gel column chromatography to obtain 8 fractions, wherein the silica gel column chromatography gradient elution comprises petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether, acetone and triethylamine is 20: 1: 0.1, 1: 0.1: 1: 5: 1: 1.5: 0.1: 1: 2, the silica gel column chromatography gradient elution is 0.5: 1: 2, the silica gel column chromatography gradient is 0.5: 1: 2, the silica gel gradient is 0.5-5, the silica gel gradient is obtained, the silica gel column chromatography gradient is used as the third, the third step, the fourth step is the third step is the fourth step, the fourth step is the third step, the third step is the third step, the fourth step is the third step, the third step is the third, the third step is the third, the third step is the third, the third is the.
The monomeric compound 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride separated from the Fritillaria pallidiflora extract prepared in example 6 of the inventionNMR spectra of (8) -propionic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Seawan-6-oxo-5 α,20 β -diol ebeine, respectively (R) (H-H1H-NMR) and nuclear magnetic resonance carbon Spectroscopy (C13C-APT).
The monomeric compounds 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propanoic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Saiwannin-6-oxo-5 α,20 β -diol ibeibening isolated from the extract of Fritillaria pallidiflora prepared in example 6 of the present invention1The H-NMR spectra are shown in FIG. 1 to FIG. 4, wherein the 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid is1H-NMR spectrum shown in figure 1, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol1The H-NMR spectrum is shown in FIG. 2, and the product is obtained from 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol1The H-NMR spectrum is shown in FIG. 3, and the H-NMR spectrum is 17 β -Sewasatin-6-oxo-5 α,20 β -diol ibeninine1The H-NMR spectrum is shown in FIG. 4. Method for separating monomeric compounds from Fritillaria pallidiflora extract13The C-APT spectra are shown in FIG. 5 to FIG. 8, wherein the 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid13The C-APT spectrum is shown in figure 5, and the C-APT spectrum is 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol13The C-APT spectrum is shown in FIG. 6, and the C-APT spectrum is 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol13The C-APT spectrum is shown in FIG. 7, and the C-APT spectrum is 17 β -Seawanine-6-oxo-5 α,20 β -diol ibeianine13The C-APT spectrum is shown in FIG. 8. Performing map analysis on fig. 1 and 5, and attributing peaks in fig. 1 and 5, wherein the peak attribution in fig. 1 and 5 is shown in table 1; performing map analysis on fig. 2 and 6, and attributing peaks of fig. 2 and 6, wherein the peak attribution of fig. 2 and 6 is shown in table 2; performing map analysis on fig. 3 and 7, and assigning the peaks in fig. 3 and 7, wherein the peak assignments in fig. 3 and 7 are shown in table 3; the peaks in fig. 4 and 8 were assigned by analyzing the maps in fig. 4 and 8, and the peak assignments in fig. 4 and 8 are shown in table 4. As is apparent from the data in tables 1, 2, 3 and 4, fraction of Fritillaria pallidiflora extract prepared in example 6 of the present inventionThe isolated monomeric compounds 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol, and 17 β -Sevannin-6-oxo-5 α,20 β -diol ebinin, wherein the chemical structural formula of 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid is shown in the specification
Figure BDA0002336294560000081
The chemical structural formula of the 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol is
Figure BDA0002336294560000091
The chemical structural formula of the 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol is shown in the specification
Figure BDA0002336294560000092
The chemical structural formula of the 17 β -Seawanine-6-oxo-5 α,20 β -diol ebeibenine is shown in the specification
Figure BDA0002336294560000093
The monomer compound separated from the Fritillaria pallidiflora extract is easy to dissolve in chloroform and methanol.
The monomeric compounds 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propanoic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -sevannin-6-oxo-5 α,20 β -diol ibeibenin isolated from the extract of fritillaria pallidii prepared in example 6 were subjected to in vitro antitumor potency experiments using MTT colorimetry.
The monomeric compounds 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Savonin-6-oxo-5 α,20 β -glycol ibebeining isolated from the Fritillaria pallidiflora extract prepared in example 6 of the present invention were used as experimental groups, Paclitaxel (Paclitaxel, antitumor drug) as a control group, and blank groups were simultaneously selected from the experimental group, the control group and the blank group as HepG-2 (human liver cancer)Cancer) cell and HeLa (human cervical cancer) cell as the subjects, diluted with the medium, and then treated at 6X 104The density of each group is inoculated in a 96-well plate, each well is 100 mu l, after the group is normally cultured in an incubator for 24 hours, the corresponding drugs are added into each group, the final concentration of each group of drugs is respectively 2.5 mu g/ml (1 group), 5 mu g/ml (2 group), 10 mu g/ml (3 group), 20 mu g/ml (4 group) and 40 mu g/ml (5 group), 5 concentrations are set, and each concentration is 3 multiple wells; after 48 hours of incubation, each well was stained with MTT 10. mu.l; continuously culturing for four hours, removing the original culture solution, adding DMSO (dimethyl sulfoxide) 150 μ l into each well, shaking on a shaking table at low speed for 10min to dissolve the crystal completely, detecting optical density value at 570nm wavelength of an enzyme linked immunosorbent assay, and calculating 50% Inhibitory Concentration (IC) according to the optical density value50μ g/mL), optical density value calculation IC50The calculation method of (2) is a prior known technology. IC of experiment group and control group on HepG-2 cell and HeLa cell50As shown in Table 5, it can be seen from the data in Table 5 that the monomeric compounds of 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol, and 17 β -Saiwannin-6-oxo-5 α,20 β -diol ibeibenin isolated from the extract of Fritillaria pallidii of the present invention have certain inhibitory effects on HepG-2 cells and HeLa cells.
In conclusion, the invention discloses the application of the monomeric compound 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Saiwanning-6-oxo-5 α,20 β -diol ebeibening separated from the extract of Fritillaria pallidiflora for the first time, wherein the monomeric compound has stronger inhibition effect on HepG-2 cells and HeLa cells, and can be used for preparing tumor prevention medicines, anti-tumor medicines and health products for preventing and treating tumors.
The technical characteristics form an embodiment of the invention, which has strong adaptability and implementation effect, and unnecessary technical characteristics can be increased or decreased according to actual needs to meet the requirements of different situations.
TABLE 1
Figure BDA0002336294560000101
TABLE 2
Figure BDA0002336294560000102
Figure BDA0002336294560000111
TABLE 3
Position of Chemical shift value (delta) of hydrogenH) Chemical shift value (delta) of carbonH)
4 4.32(2H,t,J＝6.6Hz) 29.9
5 4.14(1H,t,J＝6.6Hz) 50.5
6 4.07(2H,t,J＝6.6Hz) 65.4
2’ -- 144.5
3‘ 7.63(1H,d,J＝7.2Hz) 124.9
4’ 7.42(1H,d,J＝7.2Hz) 127.3
5‘ 7.35(1H,d,J＝7.2Hz) 127.8
6’ 7.79(1H,d,J＝7.2Hz) 120.3
TABLE 4
Figure BDA0002336294560000121
TABLE 5
Figure BDA0002336294560000122

Claims (10)

1. A monomer compound separated from Fritillaria pallidiflora extract is characterized by comprising 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid, 4- ((E) -3-hydroxybutyl-1-alkenyl) -3,3, 4-trimethylcyclohexanol, 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol and 17 β -Saiwannin-6-oxo-5 α,20 β -glycol ebeiannin, wherein the chemical structural formula of the 7- (4-hydroxy-3-methoxyphenyl) -9, 10-anhydride-8-propionic acid is shown in the specification
Figure FDA0002336294550000011
The chemical structural formula of the 4- ((E) -3-hydroxybut-1-enyl) -3,3, 4-trimethylcyclohexanol is
Figure FDA0002336294550000012
The chemical structural formula of the 2- (tetrahydro-5- (2-hydroxyphenyl) -2H-pyran-3-yl) phenol is shown in the specification
Figure FDA0002336294550000013
The chemical structural formula of the 17 β -Seawanine-6-oxo-5 α,20 β -diol ebeibenine is shown in the specification
Figure FDA0002336294550000014
2. The monomer compound separated from the Fritillaria pallidiflora extract according to claim 1 is obtained by the steps of crushing Fritillaria pallidiflora, adding methanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, recovering under reduced pressure for 1-3 hours each time, merging reflux extracting solutions each time, recovering under reduced pressure, concentrating to obtain Fritillaria pallidiflora total extract, adjusting pH to 3.5 with 2% hydrochloric acid, kneading and filtering to obtain acid solution, extracting and degreasing the acid solution with petroleum ether, adjusting pH to 10 with ammonia water, extracting with chloroform, concentrating the extracting solution to obtain chloroform extract, performing gradient elution on the chloroform extract with silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of silica gel column chromatography comprises petroleum ether, acetone and triethylamine, the volume ratio of petroleum ether, acetone and triethylamine is 20: 1: 0.1, 15: 1: 0.1, 10: 1: 0.1: 1: 0.1: 1: 1.1: 1, cyclohexanol and the gradient eluent is prepared by a step of 0.1: 1: 2, purifying the eluent with phenyl group chromatography, the gradient chromatography is prepared by a step of silica gel column chromatography, the fourth step, the steps of silica gel column chromatography is prepared by a-3: 3-2, the gradient elution is prepared by a-3, the steps of silica gel column chromatography is prepared by a-2, the steps of silica gel column chromatography is prepared by a gradient chromatography, the steps of silica gel column chromatography is prepared by a-3, the steps of silica gel column chromatography is prepared by a gradient chromatography, the steps of silica gel column chromatography is prepared by a silica gel column chromatography, the steps of silica gel column chromatography is prepared by the steps of a step of the steps of silica gel column chromatography, the steps of the.
3. The isolated monomeric compound of Fritillaria pallidiflora extract according to claim 1 or 2, wherein in the first step, 8ml to 10ml of methanol is added per 1g of Fritillaria pallidiflora.
4. The monomer compound separated from Fritillaria pallidiflora extract according to claim 1, 2 or 3, wherein in the fourth step, the fifth step, the sixth step and the seventh step, the eluent of the high performance liquid chromatography gradient elution is a mixed solution of methanol and water, and the volume ratio of methanol to water in the mixed solution is 60: 40. 70: 30. 85: 15 and 80: 20.
5. a method for preparing a monomer compound separated from an extract of Fritillaria pallidiflora is characterized by comprising the steps of firstly, crushing Fritillaria pallidiflora, adding methanol, soaking for 3-4 hours at room temperature, heating and refluxing for 3 times at 50-60 ℃, recovering under reduced pressure and concentrating to obtain a total extract of Fritillaria pallidiflora, secondly, adjusting the pH value of the total extract of Fritillaria pallidiflora to 3.5 by using 2% hydrochloric acid, kneading and filtering to obtain an acid solution, extracting and degreasing the acid solution by using petroleum ether, adjusting the pH value to 10 by using ammonia water, extracting by using chloroform, concentrating the extract to obtain a chloroform part extract, thirdly, performing gradient elution on the chloroform part of the extract by using a silica gel column chromatography to obtain 8 fractions, wherein the gradient elution of the chloroform part comprises the petroleum ether, acetone and triethylamine, the volume ratio of the petroleum ether, the acetone and the triethylamine is 20: 1: 0.1, 15: 1: 0.1, 10: 1: 0.1: 1: 1.1: 1: 1.5, the cyclohexanol and the triethylamine, purifying the extract by using a high-2-phenyl alcohol, collecting the gradient elution at the sixth, purifying the fifth step, the fourth, the third, the fourth, the fifth, the third, the fourth, the third, the fourth, the fifth, the third, the fifth, the sixth, the fifth, the sixth, the fifth, the sixth, the fifth, the sixth, the fifth, the sixth, the fifth, the sixth, the fifth, the sixth, the.
6. The isolated monomeric compound of Fritillaria pallidiflora extract according to claim 5, wherein in the first step, 8ml to 10ml of methanol is added per 1g of Fritillaria pallidiflora.
7. The monomer compound separated from Fritillaria pallidiflora extract according to claim 5 or 6, wherein in the fourth step, the fifth step, the sixth step and the seventh step, eluents for gradient elution by high performance liquid chromatography are all mixed solution of methanol and water, and the volume ratio of methanol to water in the mixture is 60: 40. 70: 30. 85: 15 and 80: 20.
8. an application of a monomer compound separated from Fritillaria pallidiflora extract in preparing medicine for preventing tumor is disclosed.
9. An application of a monomer compound separated from Fritillaria pallidiflora extract in preparing antineoplastic agent is provided.
10. An application of a monomer compound separated from Fritillaria pallidiflora extract in preparing health products for preventing and treating tumor is disclosed.
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